Recovery of dihydrostreptomycin



United States Patent RECOVERY OF DIHYDROSTREPTOMYCIN 2,950,277 Patented Aug. 23, 1960- ice drochloride. These salts are colorless or white crystalline, or powdery substances. They are odorless or nearly odorless, taste slightly bitter, and their aqueous solutions are levorotatory, and an aqueous solution of the Sueo Tatsuoka, Osaka, Tsunaharu Kusaka, Amagasaki, y l of the Sulfate hOWS ]D Akira Miyake,Nichinomiya,Michitaka Inoue, Minamiis well known that dihydrostreptomycm shows a wide kn, Osaka, Yutaka Shiraishi, lkuta-ku, Kobe, Hid sp f antibacterial spectrum against gram-positive, gram-negagg y l zil s gia lj Masahlkq lmamshti, tive and acid-fast bacteria.

.gashiyodogawa' s apan asslgnors 9 A method for the direct preparation of dihydrostrep ai g a g g Doshomachl l0 tomycin has recently been developed, and is disclosed and gas S P claimed in copending application Serial No. 647,681, Filed Mar. 21, 1957, Set. No.'647,682 filed on even date herewith (now US. Patent No.

2,931,756). Briefly stated, the said method comprises 3 Clalms' (CL 260-210) inoculating a dihydrostreptomycin-producing strain oi 5 Streptomyces on a liquid medium and cultivating the said th The f dili c l i b stii tgiif giifirgg th gg fgf strain aerobically to produce dihydrostreptomycin in an ti 6 g zi g i h bpeen girecfl reduced b the amount sufficient to be collected. The present invention g g q 1 f dih i tom cim (5min mimic? is concerned, more particularly, with recovering the di- 6 1 n o y p y p g hydrostreptomycin from the resultant fermentation broth.

In preparin the dihydrostreptomycin according to the Dihydrostreptomycm 1s a very useful substance for Z ti t f St t medical treatment because it shows a strong and wide 5 t? 5 3 i a i antibacterial spectrum like streptomycin and is, in addi- 6 mp Dye so at as 1 pro .uces y m rep 0' tion, less toxic and more stable than the latter. It is i For p a new sPecles sqjmfated by t also known that dihydrostreptomycin, which resembles 25 apphcants named m the copendmg aPPhcatlon and streptomycin in antibacterial properties, has the advanlgnfi-ted 23572 can be P f for the sald P tage over the latter that it exhibits much lower neuro T1113 51366168 has the characteristics Shown 111 P o toxicity. Dihydrostreptomycin has previously been obtable Color names marked Wlth Rdg 111 the t l tained by hydrogenation of the carbonyl radical of strep are based 011 Ridgways C0101 Standards and Nomentomycin and placed on the market as itssulfate or hy- STRAIN 23572 Cultural characteristics Medium Remarks Growth Aerial mycelium and spores Soluble pigment Czapek agar CDlOrlP e White N Glucose asparagine agar do White to Smoke-gray (Rdg XLVI, d0 Abundantly interspersed with small 2id) or Vinaceous-bufi (Rdg X1, moist black patches which gradu- 17 "-d). ally spread over the whole surface. Reverse Cream-bull (Rdg XXX, 19d) or Cartridge-buff (Rdg XXX, 19i), later becoming Chamois (Rdg XXX, 19b). Starchagar do White to Pale smoke gray (Rdg do Reverse Cream-boil (Rdg XXX,

XLVI,21""-i). 19b). Hydrolysis slight. Calcium malate agar Colorless, later becoming scanty White do Bufi-yellow (Rdg IV,

Glycerine nitrate agar Colorless do do- Dextrose nitrate agar do do do Bouillon agar do Ione do Gelatin do. ..do do Moderate liquefaction.

Potato plug wzlii te tg) Smoke gray (Rdg XLVI, ...do- Moist black patches observed.

Yeast extract agar wllite tg) Light drab (Rdg XLVI, .do Partially moistened.

Whole egg White do Milk -do do d0. Peptonization slowly.

Glycerin asperagiuate agar do lv2hli te tg) Smoke gray (Rdg XLVI, do

Peptone nitrate broth Nitrate reduction.

Aerial mycelium of this strain shows spiral, 0.8-1.2 conidia oval, 1-1.5 x 1.5-2p.

Carbon utilization of the strain No. 23572, measured by the Pridhams method, is as follows:

d(+) -Xylose l(+)-Arabinose l(+)-Rhamnose d-Fructose- Lactose d(+)-Rafifinose Salicin Inulin Na-acetate. Na-citrate.. :E Na-succinate dl-In Control No growth.

+: Poor growth.

+++: Good growth.

=l=: Growth doubtful.

++: Fair growth.

From the characteristics mentioned above, the species is manifestly anew species, and so it was named Streptomyces humidus nov. sp. Nakazawa et. Shibata.

The above mentioned Streptomyces humidus strain 7 represents only-one example of the strains usable in the prescnt invention. Strains belonging to other species also can be. used for the same purpose so far as they produce dihydrostreptomycin as a metabolite, even if 7,

they do not much resemble Streptomyces humidus' nov. sp. in properties. 7

' According to the said copending application, various substances which are used as nutrient source for cultivation of'general micro-organisms can be employed in the method'thereof. As carbon source there are used starch, lactose, sucrose, dextrin, glycerin and maltose for example. And as nitrogen source there are employed organic or inorganic, nitrogen-containing substances such as soy-bean meal, meat extract, peptone, peanut powder, casein, amino acids, yeast, bran, corn-steep liquor, cotton-seed powder, nitrates, urea and ammonium compounds. Also a small amount ofinorganic salts and growth-promoting substances may be added to the medium. As other nutrient sources there are used vmyceliaofa strain belonging to Penicillium or its culture broth for instance. Any culture media suitable for the cultivation of Streptomyces griseus may be used; A precursor alsomay be added if necessary.

v I'he culture. medium. may be solid or liquid, but an aerobic submerged .culture-ispreferable for industrial purpose.

, .When Streptomyces humidus is used as the dihydrostreptomycin-producing micro-organism and the cultivation is conducted under aerobic submerged conditions, the cultivation is preferably conducted at a temperature of about 24 to 30 C. over a period of 3 to 8 days, but the. temperature and the period must be adjusted accord ing to the other culture conditions.

The dihydrostreptomycin thus produced can be collected in an optional purity by means of various physical or chemical methods, utilizing the properties of dihydrostreptomycin.

Isolation of dihydrostreptomycinfrom a dihydrostreptomycin-containing fermentation broth or from its processed substance was accomplished for the first time by the present inventors.

Collection in this specification means to obtain dihydrostreptomycin in an optional state purer than the material in accordance with the purpose of its use.

The physical and chemical methods for purifying dihydrostreptomycin include various means, for example, those which utilize diiferences between dihydrostreptomycin or its salts and impurities in adsorbability, solubility, distribution coeflicient between two solvents and ionic bonding strength.

In the case of liquid culture the accumulation of dihydrostreptomycin reaches several ten to several thousand 'microgramsper cc., but the dihydrostreptomycin can be effectively separated even froman about 10 'y/Cc. solution.

r The material may be subjected to the above methodsafter part or most of impurities has been removed by processes such as change of pH of the composition, formation of salts, precipitation, removal of solid materials, adsorption and extraction with a solvent.

- Isolation of dihydrostreptomycin from theoulture broth of a dihydrostrcptomycin-producing micro-organism or its. processed substance has never been reported; Such; a material of course. contains various impurities, such as. carbohydrates, protein substances, salts, animal and vege table substances, mycelia of micro-organisms and theirmetabolites. Each of the impurities includm the follow ing substances:

Carbohydrate:

drates, etc.

Protein substance: amino acids, casein, meat extract, protein, peptone, etc.

' Animal and vegetable substance: protein substances, fats,

.Metabolites of micro-organism: enzymes,yprotein sub- 7 stances, sugars, polyhydric alcohols, fatty acids, etc.

Besides these substances, synthetic compounds such as urea may be present.

Thus, the composition of. impurities. inthe. material may be very complicated, but those in the pre-processed material varies in quality and quantity with the extent and kind of the process. In, the latter case, however, other impurities such as a pH-regulating agent, precipitant, adsorbent, etc. may be present. 7

In the present invention, dihydrostreptomycin is collected from the materials by various means utilizing difference between dihydrostreptomycin and the co-existing impurities in physico-chemical properties.

One of the means utilizes difierence between dihydrostreptomycin and impurities in solubility. In the liquid culture a dihydrostreptomycin solution is obtained by filtering the broth because it. accumulates preferably in the liquid part. But a minor portion of the active compound is found in thesolid part, and it may be collected by such a method as extraction'with hot water. In this case, impurities of protein series can be eliminated by adjusting pH to isoelectric point of protein or by adding materials which precipitate with the impurities, at the same time with, or before or after, the removal of the mycelia. If the pH of the fermentation broth is high, addition of an acid substance is convenient to precipitate protein more easily. If the concentration of dihydrostreptomycin in the solution issuitable, the antibiotic can be precipitated merely by adding a suitable precipitant.

When the concentration of dihydrostreptomycin is not suflicient, part of the solvent is evaporated. As precipitant are used, for example, phosphotungstic acid, helianthine, alkyl or aralkyl sulfonicacids, acid azo-compounds and polyhalophenol compounds. .They all form salts with dihydrostreptomycin. Many other precipitants may be used, and most of them are acid compounds having carboxylic radical. In some cases the salt of dihydrostreptomycin thus formed may be double or complex salt. As will be described later, an ion-exchanger also can be used as a kind of precipitant. To expedite the precipitation of the active compound or impurities, salting out may be used if necessary.

In the case of solid culture, the solid material is ex- 7 tracted with water, an aqueous solvent or other suitable solvent to obtain a solution of dihydrostreptomycin. If

starch, sugars, water-soluble carbohy- I the fermentation broth is pro-processed into a solid materral, this can be Worked up as above, but in this case the extraction 'is conducted at a suitable pH.

Another means for the separation of dihydrostreptomycin consists in utilizing differences between the active compound and impurities in adsorbability. Dihydrostreptomycin in. the culture filtrate, forexample, is ad sorbed by the addition of a suitable adsorbent. Adsorption chromatography may be used if necessary. As adsorbents there are employed, for example, active carbon, alumina and silica gel. Separation of the adsorbent leaves the greater part of impurities in the liquid portion.

. The adsorbent is then eluted with a suitable solvent, leaving non-elutable impurities in the adsorbent. The elution is generally conducted with an acid solvent. Chroma:

' .tography eifects even more exact separation.

Difference between the active compound and impuritiesin distribution coefiicient between two solvents is also utilized -for the separation. Water or an aqueous solvent and an organic solvent immiscible with the former are used as the solvents. In this case, the active assume butyl ketone, diethylether, and halohydrocarbon such as chloroform, carbon tetrachloride. This method may be conducted in the form of distribution chromatography or counter current distribution method.

The separation may also be conducted by utilizing difierence between the active compound and impurities in ionic bonding strength. This is efiected, for example, by bringing a solution of the active compound in contact with an ion-exchanger. In this case, an anion'exchanger adsorbs anionic impurities, leaving the active compound in the solution, while a cation-exchanger adsorbs the active compound. In the latter case, cationic impurities are also adsorbed, but this is minimized by using a suitable ion-exchanger. type easily adsorbs impurities, but the resin of carboxylic acid type has little such a tendency. Addition of the ion-exchanger may be effected by batch method, but use of a column of the resin is convenient in many cases. This method can be applied to a dilute solution of the active compound, such as the culture filtrate. I

Dialysis may be employed for removing impurities with large molecular weight, but this process requires complicated procedures.

The above-mentioned means are used separately or in combination according to the quality of the material and purpose of the use of the product. tions may be carried out between or before or after the above means. For example, when the material is a fermentation broth, it is filtered to remove solid materials or substances precipitated after the cultivation. The active compound in the filtrate is adsorbed in a cationexchanger and then eluted with an acid solvent. Thus a concentrated solution of the active compound containing a small quantity of impurities .is obtained. The solution is further concentrated if necessary and subjected to adsorption chromatography to eliminate almost all the impurities. On the other hand, the active compound in the concentrated solution may be precipitated with a suitable precipitant. The precipitate is then dis solved in a suitable organic solvent and an acid is added, whereupon the corresponding acid salt of dihydrostreptomycin is obtained. If the product is recrystallized, the acid salt is obtained in almost pure state.

Throughout all the means, such procedures as filtration, washing, heating, cooling, mixing, distillation, evaporation, drying, may be employed separately or in combination.

In order to prevent decomposition of the active compound, all the operations are conducted under not extremely acid or alkaline conditions. Heating may be applied if necessary, but it is preferable not to heat too long. Unlike many other antibiotics, dihydrostreptomycin is comparatively stable in the fermentation broth, so all the operations can be effected without difiiculty.

The obtained products are identical with authentic dihydrostreptomycin, or the corresponding salts. Thus, the infra-red absorptive spectrum of the sulfate of Antibiotic 23572 is in complete accord with that of authentic dihydrostreptomycin sulfate. The said products also coincide with authentic dihydrostreptomycin in all other respects, eg as to ultra-violet spectra, analytical values, chemical and physical properties, such as melting point, response to maltol reaction, response to Sakaguchi reaction, antibacterial potency to B. subtilis, chemotherapeutic elfect, antibacterial spectrum, etc. Thus e.g. the

Other opera-.

The resin of sulfonic acid sulfate of antibiotic 23572 was compared with the cordmercial dihydrostreptomycin sulfate as to weight for causing complete inhibition of test micro-organisms to give the following results:

ANTIBACTERIAL SPECTRUM A com- Sulfate of mercial Antibiotic dihydro- Micro-organism 23572. streptomegJml. mycin sulfate meg/ml Staphulaccus aureus Terajima l 1 Bacillus subtilis (POI219)- 0. 5 0. 5

Salmonella typhosa. 64 64 Sbzgella dysentma 1 1 Vibria cholerae.-- 8 8 Proteus vulqansfl 8 8 Escherichia coli 4 4 Aeperqillua niger 100 Penicillium notatum 100 100 Candida albicans 100 100 Mycobacterium 607 2. 0 2. 0 Adycobuclerium am'um 1.0 l. 0

The foregoing procedures are described in more detail in the following examples, but these are given by way of illustration and not for purposes of limitation. In these examples, the antibiotic potency is assayed by "cylinder-plate method using Bacillus subtilis (POI-219) as the test organism except when the assay method is particularly set forth. The first example sets forth a representative manner for the direct preparation of the dihydrostreptomycin according to the aforesaid copending application. The other examples are representative of embodiments of the present invention.

The single figure accompanying this specificationshows the spectrum of antibiotic 23572 (solid line) in comparison with that of dihydrostreptomycin (dotted line).

(Each spectrum was observed on its sulfate in Nujol mull, respectively.)

A Streptomyces humidus strain, e.g. No. 23572, was subjected to aerobical tank culture for 96 hours. To 700 liters of the culture filtrate (pH 8.5; potency 35 u./ cc. (dilution unit) against E. coli) was added 7 kg. (1%) of active carbon, and the mixture was stirred for 30 minutes to adsorb the active compound. The active carbon was eluted with 10 times its volume of methanolic hydrogen chloride for 30 minutes at pH 2.0 to obtain liters of the eluate (potency 100 u./ cc. (dilution unit)). The elution was repeated again to obtain ca. 70 liters of the eluate (potency 35 u./cc. (dilution unit)). The combined eluates were neutralized with N-NaOI-l to pH 6.5 and concentrated in vacuo at low temperature to about 300 cc. During the process the separated sodium chloride was removed now and then. 3 liters of anhydrous acetone was added to the concentrated solution, and the precipitated active compound was dried in vacuo to give white powder. The yield was 147 g. or 60% (potency 100 u./mg. (dilution unit)). The product was negative to maltol reaction but positive to Sakaguchi reaction.

EXAMPLE 2 Purification by ion-exchanger V (a) 500 liters of the culture filtrate (pH 7.7; potency; 350 u./cc. (dilution unit) against E. vcoli) obtained as in Example 1 was passed through a tower packed with 30 j liters of Amberlite IRC-50 (H-type) at a rate of 1.8

. (91.5%)) at low temperature under reduced pressure, removing the separated sodium chloride occasionally.

(b) To- 748 liters of the culture filtrate (content of dihydrostreptomycin, about 2200 meg/m le) obtained as inExample 1 was added 4.1 kg. of oxalic acid, and the resulting precipitate was removed by the De Laval sepav rator. The pH was adjusted to. about 7.0 with sodium. hydroxide solution and the resulting precipitate was removed again. Thesolution was then passed through a tower packed with 6.3 liters of Amberlite IRC-SO (Natype) at a rate of 40 liters per hour, whereupon the eflluent showedonly 12-6 rncg./ml. potency. The. resin was washed with water and. the active compound was eluted by passing 4% hydrochloric acid through the tower at a rate of 3' liters per hour.

20 liters of the eluate thus obtained was neutralized by addition of Amberlite IR-45 (OH-type) or- Amberlite IRA-400 (OH-type), and was concentrated to 2.5 liters (potency 368,000 mcg./cc.) at low temperature under reduced pressure.

EXAMPLE 3 Extraction with organic solvent To 1 liter of the culture filtrate (potency 100,1 meg/m1. against B. subtilis) obtained as in Example 2(b); was added 400 cc. of n-butanol (or iso-amyl alcohol) containing 3% of lauric acid, the aqueous layer was adjusted to pH 7.5 and the mixture was shaken for 2.0 minutes. After separation of the butanol (or isoj-amyl alcohol) layer the same extraction was repeated once more. The combined butanol (or iso-amyl alcohol) solutions were shaken with dilute sulfuric acid and water in such a way that the resulting aqueous layer reached 120 cc. and its pH was 2.0. After repeating the same extraction again, the combined extracts were shaken with ether to remove the lauric acid therein. The potency of the resulting solution (250 cc.) was 2891 meg/ml. against B. subtilis, or 72.5% in the yield.

EXAMPLE 4 Chromatography on active carbon 7 1.1iter of a solution obtained as in Example 2(a) (potency 160,000 u./cc. (dilution unit) against E. coli) was adjusted to pH 3.5 with N-HCl and passed through;

a tower (8 cm. in diameter, 50 cm. in height) packed- Potency against E. coli. (u. loc.). (dilution unit) Sakaguchi Fraction No. reaction The fractions. l3.2l were. cornbined ,.'ad justed to pH 6.5 by the addition of Amberlite IRA-400 (OH-type) and concentrated at low temperature under reduced pressure. To the'concentrated solution was added ten times its volume of anhydrous acetone, and the resulting precipitate wasseparated by decantation and driedto obtain l-Ol 'g. of the hydrochloride as a colorless; amorphous substance -(783.3 mcg./mg. as dihydrostreptomycin). To a solution of 10- g. of the product in 50 cc. of water was added a solution of 6 g. of MgSQ .7H O in 7 cc. of water. The mixture was adjusted to pH 6.0 with dilute sulfuric acid and filtered, and methanol was addeddropwisel'with stirring until tu'rbiditywas formed, keeping the;temperature of themixture at 5055 C. Some pieces of crystals of dihydrostreptomycin' sulfate were added to the turbid solution and left standing at '5055 C., when the solution separated crystals'soon (ca. 5 minutes) and at the same time became clear, 'Methanol was further added until the solution became a little turbid and the mixture was left standing as above. In this way, the process was repeated until amount of the methanol added reached 100 cc. andthe mixture became clear. Then the temperature of the mixture was reduced to room temperature, and the crystals were filtered and then stirred in 50% methanol for 5 minutes. The crystals were, filtered, washed with methanol and dried at 50 C. under reducedpressure. The yield was 8.9 g. (738- mcg./mg. as dihydrostreptomycin). Several recrystallizations heightened the potency to about 780 meg/mg. as dihydrostreptomycin. It was found that'in the above process, MgSO, exhibits the action to destroy histamine-like substance contained in the material.

by means of dilute hydrochloric acid, adjusting the pound dissolved, forming its sulfate.

EXAMPLE 5 5 cc. of the concentrate of the active compound obtained in Example 2(b) (potency 368,000fmcg./ml.) was .diluted' with water to 50 cc., and a warm saturated solution of methyl orange was added until no more precipitate came out. After cooling, the precipitate was filtered and recrystallized repeatedly from aqueous methalnol, whereupon 2.1 g. of the heliauthate of the active compound was obtained. A suspension of the helianthate in a small amount of water was adjusted to pH 2.0 with'dilute sulfuric acid, when the active com- The solution was filtered to remove the. separated methyl orange andtreated with a small amount oi -active carbon. To .the resulting colorless solution was added 10 timesitsvolume, of anhydrous acetone, whereupon 0-.6v g. of the. sulfate of; the active compoundseparatedout.

A specimen of strain 23572 has been filed with Institute for Fermentation, Osaka- 'IFO ),'-a Japanese culture collection, and its deposit number 'is 11 0-3520.

' A specimen of the said strain 23572 has also been deposited with the American Type Culture Collection, ATCC No. 21760.

What is claimed is:

1. A process for recovering dihydrostreptomycin as its acid salt, from a fermentation broth of Streptomyces hz miifus which comprises the following. steps: filtering the. said. fermentation broth, passing the latter through a weakly acidic ion-exchange resin, washing the resin with water, eluting the resultant adsorbed dihydrostreptokY in from; the resin with an acid, concentrating, the.

eluate, adjusting the concentrate to pH 3.5, passingjthe thusrprepared: solution through. active carbon, Ieliiti'ng the so-adsorbed diliydrostreptomycin from the active 1 carbon eluatet to pH; 6.5 concentrating the resultant solution, precipitating-the so-produced hydrochloride, dissolving the latteri'n water, treating the so-obtained aqueous s'o'lu tion of the hydrochloride with an aqueous solution of 'MgSO, adjusting the mixture to pH 6.0 with dilute sulfuric acid, and isolating the so-produced dihydrostreptomycin sulfate.

2. A process according to claim 1, wherein the precipitation of dihydrostreptomycin hydrochloride is effected by means of acetone.

3. A process according to claim 1, wherein the isolation of dihydrostreptomycin sulfate is effected by means of methanol.

References Cited in the file of this patent UNITED STATES PATENTS Langlykke, et a1. Jan. 3, 1950 Wintersteiner, et a1 Mar. 21, 1950 Babson, et a1 Sept. 12, 1950 Taylor Oct. 31, 1950 Friedman Mar. 28, 1958 Bartels Jan. 13, 1959 UNITED STATES PATENT OFFICE CERTTFTCATION 0F CORRECTION Patent No 2350,27? August 23, 1960 Sueo Tatsuoka et ale It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

In the heading to the printed specification, between lines 10 and 11, thereof, insert. Claims priority application Japan March 28, 1956 Signed and sealed this 25th day of April 1961.

(SEAL) A'itest:

ERNEST W, SWTDER DAVID L LADD Attesting @fficer Commissioner of Patents 

1. A PROCESS FOR RECOVERING DIHYDROSTREPTOMYCIN AS ITS ACID SALT FROM A FERMENTATION BROTH OF STREPTOMYCES HUMIDUS, WHICH COMPRISES THE FOLLOWING STEPS: FILTERING THE SAID FERMENTATION BROTH, PASSING THE LATTER THROUGH A WEAKLY ACIDIC ION-EXCHANGE RESIN, WASHING THE RESIN WITH WATER, ELUTING THE RESULTANT ADSORBED DIHYDROSTREPTOMYCIN FROM THE RESIN WITH AN ACID, CONCENTRATING THE ELUATE, ADJUSTING THE CONCENTRATE TO PH 3.5, PASSING THE THUS-PREPARED SOLUTION THROUGH ACTIVE CARBON, ELUTING THE SO-ADSORBED DIHYDROSTREPTOMYCIN FROM THE ACTIVE CARBON BY MEANS OF DILUTE HYDROCHLORIC ACID, ADJUSTING THE ELUATE TO PH 6.5, CONCENTRATING THE RESULTANT SOLUTION, PRECIPTATING THE SO-PRODUCED HYDROCHLORIDE, DISSOLVING THE LATTER IN WATER, TREATING THE SO-OBTAINED AQUEOUS SOLUTION OF THE HYDROCHLORIDE WITH AN AQUEOUS SOLUTION OF MGSO4, ADJUSTING THE MIXTURE TO PH 6.0 WITH DILUTE SULFURIC ACID, AND ISOLATING THE SO-PRODUCED DIHYDROSTREPTOMYCIN SULFATE. 